The ability to selectively mutate and inactivate specific genes in mammalian cells would be a valuable research tool to probe critical pathways associated with cancer and other diseases and might eventually have therapeutic applications. Oligonucleotides can bind to duplex DNA and form triple helices in a sequence- specific manner. Work funded by this grant has shown that by linking a triple helix-forming oligonucleotide (TFO) to a mutagen, the sequence specificity of triplex formation can be imparted to the action of the mutagen, and so DNA damage and thereby mutations can be directed to a specific site. TFOs conjugated to psoralen were found to produce site-specific mutations in supF reporter genes in an SV40 vector in cells, at frequencies up to 2 percent. It was also found that triplex formation, even in the absence of a tethered mutagen, can be mutagenic and that triplexes can both stimulate and inhibit cellular repair pathways. Recently, targeted mutagenesis of chromosomal genes was observed, using either DNA TFOs or oligomers composed of peptide nucleic acids (PNAs), with successful targeting of a supF gene in a recoverable, chromosomally integrated lambda vector and of the hprt gene. In this renewal application, work is proposed to further investigate chromosomal gene targeting by TFOs, using new lambda vectors containing modified supF reporter genes optimized for studying triplex-directed mutagenesis. Experiments will focus on testing the influence of triplex motif, target site transcription, cell cycle phase, and chromatin structure on chromosome accessibility and TFO-mediated targeting. A series of novel TFO modifications, including base, sugar, and backbone changes, will be provided by a team of collaborators assembled by the PI and will be tested for their effect on triplex formation, stability, and mutagenesis to identify optimal reagents for gene targeting in cells. Additional experiments will further test chromosome targeting by PNAs and will explore the utility of psoralen-PNA conjugates. The roles of DNA damage and repair in the triplex-mediated mutagenesis will be examined, to identify effective reagents for genome modification and to probe cellular pathways of triple helix DNA metabolism. A series of novel TFO- mutagen conjugates will be tested, including alkylating agents, camptothecin, and triplex-specific intercalators. The repair of triplex-associated DNA damage and of triplexes, themselves, will be investigated in human cell extracts, with an emphasis on assays to examine specific aspects of the nucleotide excision repair (NER) pathway, including repair endonuclease incisions, excision of damaged fragments, and repair-associated DNA synthesis.